Page 182 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

IncuCyte Neuronal Activity Assay
®

For the kinetic quantification of neuronal activity and functional connectivity

This protocol provides an overview of the IncuCyte® S3 Neuronal
Activity Assay methodology which uses the lentiviral based
genetically encoded orange fluorescent calcium indicator. The
assay is compatible with the IncuCyte® S3 Live-Cell Analysis
System for Neuroscience using your choice of cells and treatments.

Required materials
• IncuCyte® Neuronal Activity Analysis Software Module • For iCell GlutaNeurons:
®
(Sartorius Cat# 9600-0032) — Sodium borate solution (Thermo Fisher Cat#28341)
• IncuCyte® S3 Live-Cell Analysis System for Neuroscience — Polyethylenimine (PEI) (Sigma Cat#408727)
(Sartorius Cat# 4763) — iCell® GlutaNeurons (Cellular Dynamics Cat # R1061)
• IncuCyte® NeuroBurst Orange Lentivirus — Laminin (Sigma Cat# L2020)
(Sartorius Cat# 4736) — iCell Neural Supplement B (Cellular Dynamics Cat# M1029)
• Flat bottom 96-well tissue culture plate — N-2 Supplement (Thermo Fisher Cat#1750248)
(e.g., Corning Cat #3595) — iCell Nervous System Supplement (Cellular Dynamics Cat#
• Water, cell culture grade M1031)
• Uridine (Sigma Cat# U3003) Recommended Materials for IncuCyte Neurite Analysis
®
• 5-Fluoro-2′-deoxyuridine (Sigma Cat#F0503) • IncuCyte NeuroTrack Software Module (Sartorius Cat# 9600-
0010)
• IncuCyte® rAstrocytes (Sartorius Cat #4586)
• IncuCyte NeuroLight Orange Lentivirus Reagent
®
• BrainPhys™ Neuronal Medium (Stem Cell Technologies Cat#
05790) (Sartorius Cat# 4758)
• DMEM (Invitrogen Cat#11965)
• Fetal Bovine Serum (Sigma Aldrich Cat# F2442)
• For rat cortical neurons:
— Poly-D-Lysine (PDL) (Millipore Cat#A-003-E)
— IncuCyte® rCortical Neurons (Sartorius Cat #4753)
— NeuroBasal® Medium (Thermo Fisher Cat #21103-049)
— NeuroCult™ SM1 Neuronal Supplement
(Stem Cell Technologies Cat# 05711)
— GlutaMAX Supplement (Thermo Fisher Cat# 35050061)

General guidelines

• Due to differences in neuronal sources, media and culture • Thaw no more than 1 vial of neurons at one time. Maintain
supplements may vary. We recommend using media/ neurons in liquid nitrogen until immediately before thawing
supplements supplied by the specific cell vendor. to ensure maximal performance of the cells. Complete the
• The protocol below outlines a coating of PEI/laminin for iCell following steps of the thawing procedure in a time-efficient
®
GlutaNeurons as an example of iPSC-derived neurons and manner to facilitate optimal viability and performance.
PDL for primary rat cortical neurons. Depending on neuronal • We recommend initiating activity experiments on a Monday in
source, other preferred ECM plate coatings may be used. order to avoid weekend feedings.

180